The transforming growth factor β (TGF-β) superfamily encompasses a group of structurally related proteins. The proteins of the TGF-β family are initially synthesized as a large precursor protein which subsequently undergoes proteolytic cleavage at a cluster of basic residues approximately 110-140 amino acids from the C-terminus. The C-terminal regions, or mature regions, of the proteins are all structurally related and the different family members can be classified into distinct subgroups based on the extent of their homology. Although the homologies within particular subgroups range from 70% to 90% amino acid sequence identity, the homologies between subgroups are significantly lower, generally ranging from only 20% to 50%. The active species appears to be a disulfide-linked dimer of C-terminal fragments (Hammonds et al., 1991), although for some family members (Ling et al., 1986; Cheifetz et al., 1987), heterodimers have also been detected and appear to have different biological properties than the respective homodimers.
The TGF-β family includes Mullerian inhibiting substance (MIS) (Behringer et al., 1990), which is required for normal male sex development, Drosophila decapentaplegic (DPP) gene product, which is required for dorsal-ventral axis formation and morphogenesis of the imaginal disks (Padgett et al., 1987), the Xenopus Vg-l gene product, which localizes to the vegetal pole of eggs (Weeks et al., 1987), the activins, which can induce the formation of mesoderm and anterior structures in Xenopus embryos (Thomsen et al., 1991), and the bone morphogenetic proteins (BMPs, osteogenin, OP-1) which can induce de novo cartilage and bone formation (Sampath et al., 1990). Another member of the TGF-β superfamily, myostatin (also known as growth/differentiation factor-8 or GDF-8), is synthesized by skeletal muscle, and regulates the proliferation and differentiation of myoblasts. The members of the TGF-β family can thus influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, hematopoesis, and epithelial cell differentiation (Massague, 1987).
Because the members of the TGF-β family are involved in differentiation, the association of a particular TGF-β member with a certain characteristic in an animal and the manipulation of the expression of the gene(s) associated with that characteristic is of interest. For example, muscle mass in Belgian Blue cattle, due primarily to hyperplasia of muscle cells, is increased by approximately 20% (referred to as double-muscling) with a corresponding decrease in bone and fat mass (Shahin and Berg, 1985). Belgian Blue cattle are particularly valuable livestock as they utilize feed efficiently and give rise to a higher percentage of desirable cuts of meat (Casas et al., 1997). Double-muscling in Belgian Blue cattle was observed to be an inherited trait that was recessive because heterozygotes were normal or had only a modest increase in muscle mass. Molecular analyses found that these cattle expressed a non-functional myostatin protein (see, e.g., WO 99/02667). Another breed of cattle, the Piedmontese, and myostatin gene knock-out mice, also exhibit double-muscling (McPherron and Lee, 1997; Grobet et al., 1997; McPherron et al., 1997; Kambadur et al., 1997). Thus, myostatin has been hypothesized to regulate the amount of skeletal muscle mass in a negative manner.
To prepare transgenic species having increased muscle tissue, e.g., for livestock with high muscle and protein content, the delivery of mutated myostatin genes to animals has been proposed. See, e.g., WO 98/33887. Despite the advantages of double-muscling, double-muscled cattle often have undesirable traits. The myostatin gene becomes active during the embryonic stage, and any reduction in myostatin production causes excessive muscle development in utero, leading to larger offspring. Belgian Blue calves are generally 10-38% heavier than normal, dystocias are prevalent, requiring cesarean deliveries. These animals also exhibit abnormal reproduction due to poorly developed reproductive tracts and have other anatomical abnormalities such as macroglossia. Thus, transgenic animals having mutated myostatin genes, like Belgian Blue cattle, would likely require cesarean delivery and exhibit anatomical abnormalities, a serious burden to large animal producers. Additionally, there is public opposition to genetically engineered animals for human consumption.
Thus, what is needed is a method to prepare non-transgenic animals having increased muscle mass, preferably without an increase in fat content, as well as having acceptable performance, e.g., the animals utilize feed efficiency, and retain reproductive function and general health.